Method of immunomodulatory treatment of insulin dependent diabetes mellitus using mycobacterial cell wall compositions

ABSTRACT

The present invention relates generally to a method of immunomodulating therapy and pharmaceutical compositions useful for same. More particularly, the present invention provides a method of ameliorating the effects of autoimmune conditions. Even more particularly, the present invention contemplates a method for preventing, delaying onset of or otherwise ameliorating the effects of insulin-dependant diabetes mellitus (IDDM) by administering a cell wall subunit or a chemical or functional equivalent thereof from  Mycobacterium  or a related organism or other suitable biological source. The present invention is further directed to a pharmaceutical composition useful in preventing, delaying onset of, curing, curing in association with islet replacement or otherwise ameliorating the effects of autoimmune conditions such as IDDM or for enhancing an immune response against melanoma or other cancer comprising a cell wall subunit or a chemical or functional equivalent thereof from  Mycobacterium  or a related organism or other suitable biological source. The cell wall subunit is preferably mycolyl-arabinogalactan-peptidoglycan (MAPG) or a component thereof.

FIELD OF THE INVENTION

The present invention relates generally to a method of immunomodulatingtherapy and pharmaceutical compositions useful for same. Moreparticularly, the present invention provides a method of amelioratingthe effects of autoimmune conditions. Even more particularly, thepresent invention contemplates a method for preventing, delaying onsetof or otherwise ameliorating the effects of insulin-dependent diabetesmellitus (IDDM) by administering a cell wall subunit or a chemical orfunctional equivalent thereof from Mycobacterium or a related organismor other suitable biological source. The present invention is furtherdirected to a pharmaceutical composition useful in preventing, delayingonset of, curing, curing in association with islet replacement orotherwise ameliorating the effects of autoimmune conditions such as IDDMor for enhancing an immune response against melanoma or other cancercomprising a cell wall subunit or a chemical or functional equivalentthereof from Mycobacterium or a related organism or other suitablebiological source.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated integer or group of integers but not the exclusion of anyother integer or group of integers.

Bibliographic details of the publications numerically referred to inthis specification are collected at the end of the description.

BACKGROUND OF THE INVENTION

Insulin-dependent diabetes mellitis (IDDM) is a debilitating, chronic,cell-mediated autoimmune disease characterised by lymphocyticinfiltration of the pancreatic islets and T lymphocyte-mediated mediateddestruction of insulin-producing β cells (1, 2).

Non-obese diabetic (NOD) mice are a valuable model in studying IDDM asthey spontaneously develop the disease which has many immunological andpathological similarities to human IDDM (3, 4).

It has been previously shown that administration of Freund's completeadjuvant (CFA) or Mycobacterium bovis (Bacillus Calmette-Guerin (BCG)[3]) prevents development of diabetes in NOD mice (5, 6). However,Baxter et al (7) showed the administration of BCG, although preventingdiabetes in NOD mice, precipitated a syndrome similar to systemic lupuserythematosus (SLE), precluding its use in humans.

In accordance with the present invention, it has been shown that asubunit complex from the cell wall of Mycobacterium prevents diabetes inNOD mice without risk of precipitating SLE. The subunit complex, or oneor more of its components, are useful, therefore in immunomodulatorytherapy for autoimmune diseases and for enhancing an immune response tovarious cancers.

SUMMARY OF THE INVENTION

One aspect of the present invention contemplates a method ofimmunomodulatory therapy in a mammal said method comprisingadministering to said mammal an immunomodulating effective amount of oneor more components of the cell wall of Mycobacterium or a relatedorganism or analogous components from another biological source.

Another aspect of the present invention provides a method of preventing,delaying onset of, curing or otherwise ameliorating the effects of anautoimmune disease in a mammal said method comprising administering tosaid mammal an autoimmune-preventing effective amount of one or morecomponents of the cell wall of Mycobacterium or a related organism oranalogous components from another biological source.

Still another aspect of the present invention is directed to a method ofpreventing, delaying onset of, curing or otherwise ameliorating theeffects of insulin-dependent diabetes mellitus (IDDM) in a mammal saidmethod comprising administering to said mammal an autoimmune-preventingeffective amount of one or more components of the cell wall ofMycobacterium or a related organism or analogous components from anotherbiological source.

Yet another aspect of the present invention contemplates a method ofenhancing an immune response against melanoma or other cancer in amammal said method comprising administering to said mammal animmunomodulatory effective amount of one or more components of the cellwall of Mycobacterium or a related organism or analogous components fromanother biological source.

Still yet another aspect of the present invention contemplatesimmunomodulatory therapy in a mammal said method comprisingadministering to said mammal an immunomodulatory effective amount ofmycolyl-arabinogalactan-peptidoglycan (MAPG) or a component thereof withor without other associated cell wall components and submolecularcomponents from a Mycobacterium species such as but not limited toMycobacterium bovis or a chemical equivalent of said MAPG or of acomponent thereof.

Even yet another aspect of the present invention contemplates a methodfor isolating components of MAPG for use in a therapeutic compositionfor preventing, delaying the onset of or otherwise ameliorating theeffects of diabetes in a mammal or for use in immunomodulatory therapysaid method comprising preparing cell envelopes from a species ofMycobacterium or related organism or other suitable biological source,subjecting said cell envelopes to glycolipid removing means to removesoluble glycolipids, treating the product so obtained to break themycolic acids linkage and isolating said mycolic acids, treating theremaining complex to cleave linkage at rhamnose residue connectingarabinogalactan to the peptidoglycan backbone and separating andisolating arabinogalactan and peptidoglycan.

Another aspect of the present invention provides a composition of mattercomprising MAPG or a derivative or a component thereof or itsderivative.

Yet another aspect of the present invention relates to the use of a cellwall component of Mycobacterium in immunomodulatory therapy.

BRIEF DESCRIPTION OF THE FIGURES

In the Figures:

FIG. 1 is a diagrammatic representation of a mycobacterial cell wall.

FIG. 2 is a graphical representation showing the percentage incidence ofdiabetes over time (days) following administration of phosphate bufferedsaline (PBS).

FIG. 3 is a graphical representation showing the percentage incidence ofdiabetes over time (days) following administration of BCG.

-   □0.1 mg/mouse-   +0.2 mg/mouse-   ◯0.4 mg/mouse-   ⋄0.8, 1.6, 4 mg/mouse

FIG. 4 is a graphical representation showing the percentage incidence ofdiabetes over time (days) following administration ofN-CFAglucosaminyl-N-acetylmuramyl-L-alanyl-D-isoglutamine I.V. (1.0–10μg).

-   □1.0 μg/mouse-   +2.5 μg/mouse-   ◯5.0 μg/mouse-   ⋄10 μg/mouse

FIG. 5 is a graphical representation showing the percentage incidence ofdiabetes over time (days) following administration of lipoarabinomannan(6.25–50 μg I.V.).

-   □6.25 μg/mouse-   +12.5 μg/mouse-   ◯25 μg/mouse-   ⋄50 μg/mouse

FIG. 6 is a graphical representation showing the percentage incidence ofdiabetes over time (days) following administration ofmycolyl-arabinogalactan-peptidoglycan (0.125–1.0 mg I.V.).

-   □0.125 mg/mouse-   +0.25 mg/mouse-   ◯0.5 mg/mouse-   ⋄1.0 mg/mouse

FIG. 7 is a graphical representation showing the percentage incidence ofdiabetes over time (days) following administration of mycolic acids(13–200 μg I.V.).

-   □12.5 μg/mouse-   +50 μg/mouse-   ◯200 μg/mouse.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention contemplates a method of immunomodulatory therapyin a mammal said method comprising administering to said mammal animmunomodulating effective amount of one or more components of the cellwall of Mycobacterium or a related organism or analogous components fromanother biological source or chemical equivalents of said components.

In one aspect, the present invention is directed to a method ofpreventing, delaying onset of, curing or otherwise ameliorating theeffects of an autoimmune disease in a mammal said method comprisingadministering to said mammal an autoimmune-preventing effective amountof one or more components of the cell wall of Mycobacterium or a relatedorganism or analogous components from another biological source orchemical equivalents of said components.

In another aspect, the present invention provides a method of enhancingan immune response against melanoma or other cancer in a mammal saidmethod comprising administering to said mammal an immunomodulatoryeffective amount of one or more components of the cell wall ofMycobacterium or a related organism or analogous components from anotherbiological source or chemical equivalents of said components.

Autoimmune conditions contemplated by the present invention include butare not limited to IDDM, thyroiditis, atrophic gastritis (type A),pernicious anaemia, Addison's disease, pemphigus vulgaris, pemphigoid,multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus,discoid lupus erythematosus, haemolytic anaemia, sympathetic ophthalmia,uveitis, idiopathic thrombocytopenia, idiopathic leucopenia, primarybiliary cirrhosis, autoimmune chronic active hepatitis, ulcerativecolitis, Sjogren's syndrome, dermatomyositis, scleroderma and mixedconnective tissue disease.

Cancers contemplated for immunomodulatory therapy include but are notlimited to bladder cancer, carcinoma, melanoma amongst many others.

The present invention is hereinafter described in relation to IDDM butthis is done with the understanding that the invention extends toautoimmune diseases such as contemplated above as well as theimmunomodulatory therapy of cancers.

Accordingly, the present invention particularly contemplates a method ofpreventing, delaying onset of, curing, curing in association with isletreplacement and/or pancreas transplant or otherwise ameliorating theeffects of IDDM in a mammal said method comprising administering to saidmammal an IDDM-preventing effective amount of one or more components ofthe cell wall of Mycobacterium or a related organism or analogouscomponents from another biological source or chemical equivalents ofsaid components.

The major components of the mycobacterial cell wall are: 1)mycolyl-arabinogalactan-peptidoglycan (MAPG), a polymer which providesthe structural framework of the cell wall, 2) lipids, 3)phosphatidylinositol mannosides, and 4) lipoarabinomannan (LAM). MAPGhas three major subdomains which are: 1) peptidoglycan, 2)arabinogalactan and 3) mycolic acids.N-acetylglucosaminyl-N-acetylmuramyl-L-alanyl-D-isoglutamine (GMDP) andN-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) are peptidoglycan subunitsconsidered to be responsible for most of the adjuvant activity of CFA.

The components of the Mycobacterium cell wall or of another suitablesource or their chemical equivalents contemplated for use in preventingIDDM in mammals include MAPG or components or chemical equivalentsthereof with or without other associated cell wall components andsubmolecular components or such as but not limited to mycolate,arabinogalactan and/or peptidoglycan or derivatives or chemicalequivalents thereof (see FIG. 1). MAPG or its components may be innative or chemically synthetic form. MAPG is a complex of covalentlyattached macromolecules. Mycolic acids are covalently attached toarabinogalactan which is in turn covalently attached to peptidoglycan.Reference herein to “MAPG” includes the MAPG complex isolated fromMycobacterium or related organism or other suitable biological source orto a chemically or functionally equivalent complex as well assubmolecular components including mycolic acids, peptidoglycan orarabinogalactan or chemical or functional equivalents thereof. Thesubmolecular components may be in isolated form or in partial complexforms such as comprising mycolic acids and arabinogalactan,arabinogalactan and peptidoglycan or mycolic acids and peptidoglycan orchemical or functional equivalents thereof. A particular complex mayalso comprise, for example, mycolic acids covalently linking toarabinogalactan and this may in turn be covalently linked to a portionor derivative of peptidoglycan.

A convenient source of MAPG or its components is Mycobacterium bovis orBCG. The present invention, however, extends to MAPG or its componentsfrom any species of Mycobacterium or from physiologically, genetically,biochemically or structurally related microorganisms. Examples ofsimilar organisms include Actinomycetes, Nocardia and Corynebacterium. Asimilar molecule or natural complex or components thereof may also beisolatable for other biological sources such as plants and coral. TheMAPG complex or its components may be isolated from mycobacterial cellenvelopes prepared, for example, by the method of Azuma et al (8).

According to this aspect of the present invention, there is provided amethod for isolating components of MAPG for use in a therapeuticcomposition for preventing, delaying the onset of or otherwiseameliorating the effects of diabetes in a mammal, said method comprisingpreparing cell envelopes from a species of Mycobacterium or relatedorganism or other suitable biological source, subjecting said cellenvelopes to glycolipid removing means to remove soluble glycolipids,treating the product so obtained to break the mycolic acids linkage andisolating said mycolic acids, treating the remaining complex to cleavelinkage at rhamnose residue connecting arabinogalactan to thepeptidoglycan backbone and separating and isolating arabinogalactan andpeptidoglycan.

The soluble glycolipids are conveniently removed by repeatedcentrifugation in the presence of sodium dodecyl sulphite (SDS). Theresulting insolvent envelope MAPG complex is then subjected tofractionation.

The mycolic acids linkage is preferably cleaved by saponification,base-catalysed methanolysis or ammonolysis. The remaining complex ofarabinogalactan and peptidoglycan is preferably then subjected to aSmith degradation comprising periodate followed by borohydride reductionand mild acid treatment.

The MAPG complex or its component parts or derivatives thereof may be inany convenient form such as vacuum-dried, powder, liquid or slurry.

The present invention further contemplates a composition of mattercomprising MAPG or a derivative thereof or a component thereof or itsderivative or chemical equivalents of MAPG or its components. Thesecomponents are referred to herein as “active ingredients”.

Preferably, the composition is a pharmaceutical composition for use inpreventing, delaying onset of, curing, curing in association with isletreplacement or otherwise ameliorating the effects of IDDM in mammals orfor enhancing an anti-tumour immune response in mammals. Thepharmaceutical composition may additionally comprise one or morepharmaceutically acceptable carriers and/or diluents.

According to this and other aspects of the present invention, preferredmammals include humans, primates, livestock animals (eg. cows, horses,sheep, pigs, donkeys), laboratory test animals (eg. mice, rabbits,guinea pigs, hamsters), companion animals (eg. dogs, cats) and captivewild animals (eg. kangaroos, foxes, deer).

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions (where water soluble) and sterile powders for theextemporaneous preparation of sterile injectable solutions. The presentinvention also contemplates administration via topically appliedcompositions where molecules are used to permit entry via the skin. Itmust be stable under the conditions of manufacture and storage and mustbe preserved against the contaminating action of microorganisms such asbacteria and fungi.

The carrier may be a solvent medium containing, for example, water,ethanol, polyol (for example, glycerol, propylene glycol and liquidpolyethylene glycol and the like) or suitable mixtures thereof andvegetable oils. The preventions of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example,aluminum-monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and the freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from previously sterile-filtered solutionthereof.

When the active ingredients are suitably protected they may be orallyadministered, for example, with an inert diluent or with an assimilableedible carrier, or it may be enclosed in hard or soft shell gelatincapsule, or it may be compressed into tablets, or it may be incorporateddirectly with food material (including solid or liquid products). Fororal therapeutic administration, the active compound may be incorporatedwith excipients and used in the form of ingestible tablets, buccaltablets, troches, capsules, elixirs, suspensions, syrups, wafers, andthe like. Such compositions and preparations should contain at least 1%by weight of active compound. The percentage of the compositions andpreparations may, of course, be varied and may conveniently be betweenabout 5 to about 80% of the weight of the unit. The amount of activecompound in such therapeutically useful compositions is such that asuitable dosage will be obtained. Preferred compositions or preparationsaccording to the present invention are prepared so that an oral dosageunit form contains between about 0.1 μg and about 2000 mg of activecompound. Other ranges contemplated herein include from about 1 μg toabout 1000 mg, from about 10 μg to about 100 mg and from about 100 μg toabout 50 mg. Effective amounts may also be provided as an amount perkilogram of body weight of the recipient. For example, from about 0.01ng to about 10,000 mg/kg body weight or may be administered from about0.1 ng to about 500 mg/kg body weight.

The tablets, troches, pills, capsules and the like may also contain thecomponents as listed hereafter: a binder such as gum, acacia, cornstarch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; a lubricant such as magnesium stearate; and a sweeteningagent such a sucrose, lactose or saccharin may be added or a flavouringagent such as peppermint, oil of wintergreen, or cherry flavouring. Whenthe dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both. A syrup or elixir may contain the activecompound, sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavouring such as cherry or orange flavour. Ofcourse, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compound(s) may be incorporated intosustained-release preparations and formulations.

The present invention also extends to forms suitable for topicalapplication such as creams, lotions and gels.

Pharmaceutically acceptable carriers and/or diluents include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, use thereof in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Parental compositions may be administered by, for example, intravenous(IV), subcutaneous (SC) or intramuscular (IM) routes amongst otherroutes. Dosage unit form as used herein refers to physically discreteunits suited as unitary dosages for the mammalian subjects to betreated; each unit containing a predetermined quantity of activematerial calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the novel dosage unit forms of the invention are dictated by anddirectly dependent on (a) the unique characteristics of the activematerial and the particular therapeutic effect to be achieved, and (b)the limitations inherent in the art of compounding such an activematerial for the treatment of disease in living subjects having adiseased condition in which bodily health is impaired as hereindisclosed in detail. Other routes of administration are alsocontemplated by the present invention including intracerebral,intraperitoneal, intranasal, buccal, rectal, implant, infusion,inhalation administration in addition to intravenous drip.

The principal active ingredient is compounded for convenient andeffective administration in effective amounts with a suitablepharmaceutically acceptable carrier in dosage unit form as hereinbeforedisclosed. A unit dosage form can, for example, contain the principalactive compound in amounts ranging from about 0.1 μg to about 2000 mg.Expressed in proportions, the active compound is generally present infrom about 0.5 μg to about 2000 mg/ml of carrier. In the case ofcompositions containing supplementary active ingredients, the dosagesare determined by reference to the usual dose and manner ofadministration of the said ingredients.

The active ingredients of the present invention may be administeredalone or in combination with other therapeutic molecules such asmolecules which reduce effects of the autoimmune pathology associatedwith IDDM. Alternatively, the active ingredients may be administeredwith anti-cancer agents or functionally unrelated but neverthelessuseful molecules such as antibodies, analogues or the like. A singledose may be administered or multiple doses may be required withintervals of from minutes to hours, daily to weekly or monthly toyearly.

Reference herein to “preventing” IDDM includes total prevention of IDDMor substantial prevention for a limited time (eg. from about 1 to about10 years) or delaying onset of IDDM or reducing the severity orotherwise ameliorating the effects of IDDM.

The present invention further contemplates use of a cell wall componentof Mycobacterium in immunomodulatory therapy. Preferably, the therapy isthe treatment of an autoimmune disease such as diabetes. Alternatively,the therapy is the treatment of melanoma, bladder cancer or othercancer, preferably by enhancing an immune response.

The present invention is further described by the following non-limitingExamples.

EXAMPLE 1 Components of Mycobacterial Cell Wall

Components of the mycobacterial cell wall are shown in FIG. 1.

EXAMPLE 2 Mice

Female NOD/Lt//Arc mice were obtained from the Animal Resources Centre(Canning Vale, WA, Australia) and maintained in clean conditions in theCentenary Institute Animal House. Sentinel mice were tested by serologyat four-monthly intervals for the following pathogens: mouse hepatitisvirus, rotavirus, ectomclia, mouse cytomegalovirus, polyoma virus,murine adenovirus, lymphocytic choriomeningitis virus, mouse pneumoniavirus, retrovirus, Sendai virus, Theiler's murine encephalitis virus,Bacillus piliformis, Mycoplasma pulmoniso, Bordetella bronchiseptica,Corynebacterium kutscheri, Klebsiella species, Pasturella multocide,Pasturella pneumotropica, Pseudomonas aeruginosa, Straphylococcusaureus, Streptococcus pneumoniae, Citrobacter freundii and salmonellaspecies. No mice tested positive for any of these pathogens. Mice werehoused at 21 C and 40% humidity and were fed Barastock mouse chow(Melbourne, VIC, Australia) and acidified water ad libitum.

Under these conditions, about 75% of female NOD mice spontaneouslydeveloped IDDM by 35 weeks of age. The disease process involved aprogressive preclinical phase of islet destruction which commenced at4–6 weeks of age, and concluded with the onset of clinical diabetesbetween 14 and 35 weeks of age. Within a population, disease onsetoccurred in a sigmoidal fashion with the peak incidence of IDDM at 22weeks of age, and a plateau at 35 weeks after which few previouslyunaffected mice ever progressed to diabetes (9).

NOD mice intravenously injected with a single dose of 1.0–4.0 mg ofheat-killed BCG did not become diabetic, but developed a lupus-likedisease characterised by haemolytic anaemia (indicated by a loweredhaematocrit and positive Coombs' test), increased titres of anti-nuclearantibodies (demonstrated by immunofluorescence of HEp-2-cells) andglomerulonephitis (demonstrated by immunofluorescence of C3c bound tothe renal glomeruli) (7).

EXAMPLE 3 BCG Therapy

Evans (Langhurst, UK) or CSL (Parkville, Australia) live freeze driedand attenuated Mycobacterium bovis (bacillus Calmette-Gucrin; BCG)vaccine was dissolved in isotomic saline and heat inactivated at 65 Cfor 45 minutes.

EXAMPLE 4 Mycobacterial Subfractions

Mannose-capped lipoarabinomannan (ManLAM) andmycolyl-abarbinogalactan-peptidoglycan complex (MAPG) may be prepared aspreviously described (8).N-acetylglucosaminyl-N-acetylmuramyl-L-alanyl-D-isoglutamine (GMDP) waspurchased from a commercial source (GERBU Biotechnik, Gaibery, Germany).The Applicant acknowledges with appreciation receiving samples of ManLAMand MAPG from the Tuberculosis Repository by Drs P. J. Brennan and

J. T. Belisle through NIAD13 November, 1996, NIH [Contract No.NO1-AU1-25147]. 0.

EXAMPLE 5 Random Blood Glucose Estimations

Each mouse was bled by retro-orbital venepuncture of 100–150 μl and theserum glucose concentration measured by the glucose oxidase technique ona Glucostix reagent strip (Ames, Basingstoke, UK). A mouse wasconsidered to be diabetic if it was found to have a random blood glucoselevel >11.1 mMol/l.

EXAMPLE 6 Haematocrit Measurement

Seventy-five microlitres of blood were drawn up into a heparinizedcapillary tube (Becton Dickinson, Franklin Lakes, N.J., USA) andcentrifuged at 1000 g for 15 minutes. The height of the column of packedred cells was divided by the total height of the column of blood andexpressed as a percentage.

EXAMPLE 7 Direct Coombs Test

Mice were bled and the plasma removed. Ten microlitres of packed cellswere resuspended in 5 ml PBS with 0.3% w/v bovine serum albumin, washedand resuspended in 1 ml of the same solution. Triplicates of 100 μlaliquots were placed in 96 well round bottom plates (Nunc, Denmark) with3 serial ½ dilutions. The plates were vortexed and incubated at 37 C for1 hr. Wells were then assessed for false positive results. Tenmicrolitres of 10 μg/ml polyclonal goat anti-mouse IgG (Sigma ChemicalCompany, MO, USA) added. Plates were then vortexed gently and incubatedat 37 C for a further two hours. Wells in which the cells collected in abutton were recorded as negative, while those in which the cellsremained spread of the surface over the surface of the well wererecorded as positive.

EXAMPLE 8 Assessment of Antinuclear Antibodies

Sera were assessed for binding to HEp-2 slides (Quantafluor, Chaska,Minn., USA). Slides were incubated in phosphate buffered saline (PBS)for 10 minutes. Sera diluted in PBS (starting concentration 1:100) wereincubated on the slides at room temperature (RT) for 30 minutes in amoist chamber. Slides were then washed 3 times for 5 minutes in PBS andincubated for 30 minutes at RT with 1:50 FITC conjugated rat anti-mouseIg (Serotec, Oxford, UK). Slides were again washed 3 times for 5 minuteswith PBS, mounted and examined on an Axiophot fluorescence microscope(Zeiss, DDR). Sera from MRL/lpr-lpr and BALB/c mice were used ascontrols.

EXAMPLE 9 Detection of Glomerular Immune Complex Deposits

Kidneys were embedded in Tissue-Tek OCT Compound (Miles, Elkhart, IN)and frozen for histological analysis. Sections of 6 μm were cut on aMicrom cryostat (Waldorf, Germany) and mounted on microscope slides, airdried, acetone fixed for 10 minutes and stored at −80 C in an air-tightbag containing silica desiccant. When slides were stained, they werethawed to room temperature, fixed in acetone for a further five minutesand blocked with 4% v/v foetal calf serum (FCS; CSL, Melbourne, VIC,Australia). Sections were stained with Goat anti-mouse C3c polyclonalIgG (Nordic Immunological Laboratories, Tilburg, Holland) at a 1:10dilution in PBS for 45 minutes. Slides were then washed three times inPBS, and coverslipped with polyvinyl alcohol mounting media (SanofiDiagnostics Pasteur Inc., Chaska, Minn.) and examined on a Leicafluorescence microscope (Leica Mikoskopie, Postfach, Germany).

EXAMPLE 10 Effects of Heat Killed BCG on Incidence of Diabetes

Varying concentrations of heat killed Mycobacterium bovis (BCG) wereadministered intravenously to NOD mice at 0.1 mg, 0.2 mg, 0.4 mg, 0.8mg, 1.6 mg, 4 mg relative to a control of phosphate buffered saline(PBS; FIG. 2). No diabetes was detected in mice given 0.8, 1.6 or 4 mgof BCG (FIG. 3). Doses of 0.1 mg, 0.2 mg and 0.4 mg were not completelyeffective although they did provide some protection.

EXAMPLE 11 Effects of Mycobacterial Cell Wall Components on Incidence ofDiabetes

In order to attempt to separate the activity of BCG which prevented IDDMfrom that which precipitated lupus, the mycobacterial subfractions GMDP,MAPG and ManLAM were tested for these activities. GMDP (10, 5.0, 2.5 and1.0 μg), ManLAM (50, 25, 12.5 and 6.25 μg), and MAPG (1.0, 0.5, 0.25 and0.125 mg) were each suspended in saline and injected intravenously intoeight week old female NOD mice. These doses of MAPG and ManLAM werebased on estimates of the equivalent quantities in 1 mg of BCG, whilethe does of GMDP was based on that used for immunoadjuvant activity; 10μl g being the maximum dose tolerated by mice. Groups of 5 mice weretested and the incidence of diabetes compared to 17 saline treated and14 BCG treated control mice. The results are shown in FIGS. 2 to 7.

While 10/17 (59%) PBS treated mice (FIG. 2), 12/20 (60%) of GMDP treatedmice (FIG. 4) and 13/18 (72%) of ManLAM treated mice developed diabetes(FIG. 5) no mice receiving MAPG did so (FIG. 6). Haematocrits andCoombs' tests indicated that no mice receiving MAPG developed haemolyticanaemia whereas 11/14 (79%) of BCG treated (0.8–1.6 mg) control mice hada haematocrit below 46% and 7/14 mice were Coombs' test positive.Similarly there was no increase in the expression of antinuclearantibodies in the MAPG treated mice. The effects of mycolic acidsadministration on the incidence of diabetes is shown in FIG. 7.

EXAMPLE 12 Effects of Mycobacterial Cell Wall Components on Incidence ofDiabetes

MAPG was injected intravenously into 8 week old NOD mice in dosesranging from 1 mg to 0.125 mg/mouse. All doses tested prevented IDDM.MAPG treated mice were tested for haematocrit, Coombs' test, antinuclearantibodies (ANA) and C3c complement component deposition in the renalglomeruli. No mice developed haemolytic anaemia as detected by loweredhaematocrit and positive Coombs' test. ANA levels were not raised abovethose detected in PBS treated mice. Mild C3c deposition was found in aminority of mice. It was concluded that MAPG administration preventedIDDM without inducing lupus in NOD mice.

It is significant that while MAPG was still effective at preventing IDDMwhen used at a dose of 0.125 mg/mouse, BCG was ineffective at this dose.This indicates that it is likely that the effect of the MAPG preparationused in these experiments is due to MAPG itself and not to a contaminantfrom BCG.

EXAMPLE 13 Purification of Components of MAPG

Mycobacterium cell envelopes maybe prepared by the method of Azuma et al(8) and the soluble glycolipids removed by repeated centrifugation inthe presence of SDS. The insoluble envelope component, the peptidoglycanconjugated with mycolic acids-substituted arabinogalactan, isfractionated into its constituent domains by the following procedure.

-   1. the mycolic acids linkage is cleaved by saponification,    base-catalysed methanolysis or ammonolysis and separated from the    insoluble residue.-   2. the residue is submitted to Smith degradation (periodate,    followed by borohydride reduction and mild acid treatment) to cleave    the linkage at the rhamnose residue connecting the galactan to the    peptidoglycan backbone.

Due to the nature of the furanosyl linkages in the galactan and arabinandomains, these residues are not affected by this treatment. The solublearabinogalactan is separated from the peptidoglycan by centrifugation.

EXAMPLE 14 Testing Components of MAPG

The previous example shows the effectiveness of MAPG in preventingdevelopment of diabetes relative to other components of themycobacterial cell wall. The individual constituents of MAPG arepurified according to Example 13 and tested at varying concentrations inNOD mice. The incidence of diabetes is then determined over time.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

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1. A method of immunomodulatory treatment of insulin-dependent diabetesmellitus (IDDM) in a mammal said method comprising administering to saidmammal an immunomodulating effective amount of mycolyl arabinogalactanpeptidoglycan (MAPG), isolated from the cell wall of Mycobacterium. 2.The method according to claim 1, wherein said MAPG is administered withone or more of mycolic acids, peptidoglycan or arabinogalactan.
 3. Themethod according to claim 1, wherein said MAPG is from Mycobacteriumbovis.
 4. The method according to claim 1, wherein the mammal is ahuman.